AMPKalpha2 deletion causes aberrant expression and activation of NAD(P)H oxidase and consequent endothelial dysfunction in vivo: role of 26S proteasomes

Abstract

Rationale: AMP-activated protein kinase (AMPK) is an energy sensor and ubiquitously expressed in vascular cells. Recent studies suggest that AMPK activation improves endothelial function by counteracting oxidative stress in endothelial cells. How AMPK suppresses oxidative stress remains to be established.

Objective: The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress, and endothelial function.

Methods and results: The markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91(phox), p47(phox), p67(phox), NOX1 to -4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IkappaBalpha degradation, and nuclear translocation of nuclear factor (NF)-kappaB (p50 and p65) were examined in cultured human umbilical vein endothelial cells and mouse aortas isolated from AMPKalpha2 deficient mice. Compared to the wild type, acetylcholine-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKalpha2(-/-) mice. Further, pretreatment of aorta with either superoxide dismutase (SOD) or tempol or apocynin significantly improved acetylcholine-induced endothelium-dependent relaxation in AMPKalpha2(-/-) mice. Analysis of aortic endothelial cells from AMPKalpha2(-/-) mice and human umbilical vein endothelial cells expressing dominant negative AMPK or AMPKalpha2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91(phox), p47(phox), p67(phox), NOX1 and -4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IkappaBalpha degradation, and nuclear translocation of NF-kappaB (p50 and p65), whereas AMPK activation by AICAR or overexpression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKalpha2 in low-density lipoprotein receptor knockout (LDLr(-/-)) strain markedly increased 26S proteasome activity, IkappaB degradation, NF-kappaB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, and endothelial dysfunction, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor.

Conclusions: We conclude that AMPKalpha2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way, AMPK maintains the nonatherogenic and noninflammatory phenotype of endothelial cells.

Figure 1. AMPKα2 deletion induces endothelial dysfunction in an NAD(P)H oxidase- and ROS- dependent manner

Ach-induced endothelium-dependent relaxation in WT and AMPKα2^−/− aortas after 12 h incubation with (A) tempol (10 µM), (C) PEG-SOD (100 Units/ml), and (D) apocynin (100 µM). (B & E) SNP-induced endothelium-independent relaxation in WT or AMPKα2^−/− aortas. *P<0.05 vs. WT, ^#P<0.05 vs. AMPKα2^−/−, two-way ANOVA followed by student's t-test. n=6. (F) Western blot analysis of aortic NAD(P)H oxidase subunits (p47^phox, p67^phox, and gp91^phox), phosphorylated eNOS at Ser1177 (peNOS), and total eNOS expressions in WT and AMPKα2^−/− mice. The blot is a representative of blots obtained with five mice. *P<0.05 vs. WT. (G) NAD(P)H oxidase activity in WT and AMPKα2^−/− mice aortas. *P<0.05 vs. WT. n=5. (H) Superoxide production in aortas from WT and AMPKα2^−/− mice, as assessed by DHE/HPLC. Aortas were incubated with allopurinol (50 µM), tempol (10 µM), or apocynin (100 µM) for 24 h. *P<0.05 vs. WT, ^#P<0.05 vs. AMPKα2^−/− control. n=5.

Figure 2. AICAR inhibits the expression of the NAD(P)H oxidase subunits, p47^phox and p67^phox, in HUVECs in an AMPKα2-dependent manner

Western blot analysis of p47^phox and p67^phox expression in HUVECs (A) treated with AICAR (2 mM) for the indicated times or (B) treated with varying concentrations of AICAR for 24 h. The blot is a representative of blots obtained in three independent experiments. *P<0.05 vs. control. AICAR-induced changes in p47^phox and p67^phox expression were also examined in HUVECs infected with (C & D) ad-GFP or AMPKα2-DN or (E & F) control siRNA or AMPKα2 siRNA. 24 h after infection/transfection, cells were incubated with AICAR (2 mM) or vehicle for 8 h. The blots are representative of blots obtained from three independent experiments. *P<0.05 vs. control; ^#P<0.05 vs. GFP or control siRNA; ^$P<0.05 vs. GFP or control siRNA plus AICAR. (D & F) NAD(P)H oxidase-dependent superoxide production in AMPKα2-DN-infected or AMPKα2 siRNA-transfected HUVECs. Superoxide production was assayed in HUVECs with/without apocynin (100 µM, 24 h) by DHE/HPLC, as decribed in Methods. *P<0.05 vs. GFP or control siRNA, ^#P<0.05 vs. ad-AMPK-DN or AMPKα2 siRNA. n=3.

Figure 3. AMPKα2 deletion increases NAD(P)H oxidase expression via NF-κB activation in MAECs

Lysates from WT and AMPKα2^−/− MAECs were subjected to western blot analysis for (A) p47^phox, p67^phox, and gp91^phox, as well as (B) IκBα and phosphorylated IκBα. The blots are representative of blots obtained from three independent experiments. *P<0.05 vs. WT. (C) Western blot analysis of the NF-κB subunits, p50 and p65, in nuclear fraction of MAECs (n=3). *P<0.05 vs. WT. (D) The expressions of p47^phox and p67^phox in AMPKα2^−/− MAECs treated with an NF-κB translocation inhibitor (peptide sequence: AAVALLPAVLLALLAPVQRKRQKLMP, 50 mg/ml) for 24 h (n=3). *P<0.05 vs. control. (E) p47^phox and p67^phox expressions in MAECs (WT and AMPKα2^−/−) incubated with the proteasome inhibitor, MG132 (0.5 µM), for 4 h (n=3). *P<0.05 vs. WT control, ^#P<0.05 vs. AMPKα2^−/− control.

Figure 4. Activation of AMPK by AICAR increases IκBα protein levels

(A) Western blot analysis of IκBα in HUVECs treated with AICAR (2 mM) for the indicated time. The blot is a representative of blots obtained from three independent experiments. *P<0.05 vs. control. (B) Genetic inhibition of IκBα by transfection of IκBα-specific siRNA abolishes AICAR-induced reduction of p47^phox and p67^phox in HUVECs. The blot is a representative of three individual experiments. (C) IκBα protein levels in MAECs (WT and AMPKα2^−/−) incubated with AICAR (2 mM) for 6 h (n=3). *P<0.05 vs. WT control.

Figure 5. AMPK activation suppresses 26S proteasome activity

(A) 26S proteasome activity in WT and AMPKα2^−/− MAECs treated with or without AICAR (2 mM) for 8 h (n=3) proteasome activity was assayed using fluorescent proteasome substrates. *P<0.05 vs. WT without AICAR, ^#P<0.05 vs. WT plus AICAR. 26S proteasome activity was also measured in HUVECs treated with (B) AICAR (2 mM) or metformin (2 mM) for 8 h (n=3) or with (C) AICAR (2 mM) for the indicated times (n=3). *P<0.05 vs. control. (D) 26S proteasome activity in HUVECs infected with Ad-GFP or Ad-AMPK-DN or ad-AMPK-CA for 24 h. *P<0.05 vs. GFP. n=3.

Figure 6. Chronic administration of MG132 suppresses AMPKα2 deletion-enhanced oxidative stress, in vivo

(A) Representatives of immunohistochemical staining and quantifications for 3-NT, MDA, and HNE positive proteins (original magnification ×400) in aortas from LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice. *P<0.05 vs. LDLr^−/− control mice, ^#P<0.05 vs. LDLr^−/−/AMPKα2^−/− control mice. (B) The ROS productions were detected by DHE/HPLC in LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice aortas. *P<0.05 vs. LDLr^−/− control mice. (C) The levels of 3-NT were measured by Western blot. *P<0.05 vs. LDLr^−/− control mice, ^#P<0.05 vs. LDLr^−/−/AMPKα2^−/− control mice. (D) Effects of MG132 on endothelium-dependent vasorelaxation in LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice aortas. *P<0.05 vs. LDLr^−/− control mice, ^#P<0.05 vs. LDLr^−/−/AMPKα2^−/− control mice. n=5–6 per group.

Figure 7. MG132 abrogates AMPKα2 deletion-increased expression of NAD(P)H oxidase subunits and NAD(P)H oxidase activity, in vivo

(A) Representatives of immunohistochemical staining and quantifications for NOX4, p47^phox, and p67^phox in the aortas of LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice (original magnification ×400). *P<0.05 vs. LDLr^−/− control mice, ^#P<0.05 vs. LDLr^−/−/AMPKα2^−/− control mice. (B) Western blot analysis of NOX4 and p47^phox in LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice aortas. *P<0.05 vs. LDLr^−/− control mice, ^#P<0.05 vs. LDLr^−/−/AMPKα2^−/− control mice. n=4 mice in each group. (C) Quantitative analysis of NAD(P)H oxidase activity in LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice aortas. *P<0.05 vs. LDLr^−/− ND control mice, ^#P<0.05 vs. LDLr^−/− WD control mice, ^$P<0.05 vs. LDLr^−/−/AMPKα2^−/− WD control mice. n=4 mice in each group.

Figure 8. Chronic administration of MG132 attenuates AMPKα2 deletion-induced reduction of IκBα and NF-κB transactivation, in vivo

(A) Representatives of immunohistochemical staining and quatifications for IκBα and P-p65 in aortas of LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice (original magnification ×400). *P<0.05 vs. LDLr^−/− control mice, ^#P<0.05 vs. LDLr^−/−/AMPKα2^−/− control mice. (B) Western blot analysis of IκBα in LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice aortas. *P<0.05 vs. LDLr^−/− control mice, ^#P<0.05 vs. LDLr^−/−/AMPKα2^−/− control mice. n=4 mice in each group. (C) Western blot analysis of nucleus-localized p65 and P-p65 proteins in LDLr^−/− and LDLr^−/−/AMPKα2^−/− mice aortas. *P<0.05 vs. LDLr^−/− control mice, ^#P<0.05 vs. LDLr^−/−/AMPKα2^−/− control mice. n=4 mice in each group.